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Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast.

Tapia H, Morano KA - Mol. Biol. Cell (2009)

Bottom Line: Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation.The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele.Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, TX 77030, USA.

ABSTRACT
The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Delta cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the alpha/beta importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

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Hsp90 nuclear accumulation is a consequence of glucose depletion. (A) Quiescent phase cells bearing the Hsp82-GFP fusion were shifted to fresh YPD growth medium, and the culture was followed for a growth period of 24 h. Samples were harvested at the indicated time points and processed for photomicroscopy or determination of ethanol (triangles) and glucose (circles) levels as described in Materials and Methods.
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Figure 2: Hsp90 nuclear accumulation is a consequence of glucose depletion. (A) Quiescent phase cells bearing the Hsp82-GFP fusion were shifted to fresh YPD growth medium, and the culture was followed for a growth period of 24 h. Samples were harvested at the indicated time points and processed for photomicroscopy or determination of ethanol (triangles) and glucose (circles) levels as described in Materials and Methods.

Mentions: We next analyzed the kinetics of Hsp90 accumulation into the nucleus during growth in more detail. Quiescent cultures of Hsp82-GFP cells displaying nuclear accumulation were diluted into fresh medium and allowed to resume vegetative growth at 30°C (Figure 2A, time 0). Within three hours after the shift to glucose-rich media, Hsp90 was seen to redistribute throughout the cell (a representative image from each sample is shown). Glucose and ethanol concentrations were monitored as growth continued over 24 h. After ∼18 h, glucose was nearly exhausted from the media as determined by enzymatic assay, and ethanol produced during fermentation had reached its maximum. Coincident with this, Hsp90 was again observed to accumulate in the nucleus. Hsp90 therefore seems to respond to events connected to glucose depletion rather than the complete absence of a carbon source because at 18 h ethanol was still abundant (0.5%). Together, these data suggest that the Hsp90 chaperone in yeast cells responds to progression through the diauxic shift into the quiescent state by translocating to the nucleus. However, the precise signaling events required to initiate this change in subcellular localization are likely complex as acute depletion of glucose or other growth-limiting nutrients was unable to elicit the same response.


Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast.

Tapia H, Morano KA - Mol. Biol. Cell (2009)

Hsp90 nuclear accumulation is a consequence of glucose depletion. (A) Quiescent phase cells bearing the Hsp82-GFP fusion were shifted to fresh YPD growth medium, and the culture was followed for a growth period of 24 h. Samples were harvested at the indicated time points and processed for photomicroscopy or determination of ethanol (triangles) and glucose (circles) levels as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801720&req=5

Figure 2: Hsp90 nuclear accumulation is a consequence of glucose depletion. (A) Quiescent phase cells bearing the Hsp82-GFP fusion were shifted to fresh YPD growth medium, and the culture was followed for a growth period of 24 h. Samples were harvested at the indicated time points and processed for photomicroscopy or determination of ethanol (triangles) and glucose (circles) levels as described in Materials and Methods.
Mentions: We next analyzed the kinetics of Hsp90 accumulation into the nucleus during growth in more detail. Quiescent cultures of Hsp82-GFP cells displaying nuclear accumulation were diluted into fresh medium and allowed to resume vegetative growth at 30°C (Figure 2A, time 0). Within three hours after the shift to glucose-rich media, Hsp90 was seen to redistribute throughout the cell (a representative image from each sample is shown). Glucose and ethanol concentrations were monitored as growth continued over 24 h. After ∼18 h, glucose was nearly exhausted from the media as determined by enzymatic assay, and ethanol produced during fermentation had reached its maximum. Coincident with this, Hsp90 was again observed to accumulate in the nucleus. Hsp90 therefore seems to respond to events connected to glucose depletion rather than the complete absence of a carbon source because at 18 h ethanol was still abundant (0.5%). Together, these data suggest that the Hsp90 chaperone in yeast cells responds to progression through the diauxic shift into the quiescent state by translocating to the nucleus. However, the precise signaling events required to initiate this change in subcellular localization are likely complex as acute depletion of glucose or other growth-limiting nutrients was unable to elicit the same response.

Bottom Line: Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation.The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele.Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, TX 77030, USA.

ABSTRACT
The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Delta cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the alpha/beta importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

Show MeSH
Related in: MedlinePlus